Stem Cell Artificial Meat : Industry Analysis and Forecast 2020 2025 By Key Players-, Beyond Meat, Impossible Foods, Memphis Meats, Boca Foods -…

Report Hive Research adds a new research study titled Stem Cell Artificial Meat Market report to its market intelligence database. This research report is a product of profound market analysis by our team of Research analysts, who are endowed with excellent research skills and years of experience. Our reports are majorly focused on, future trends, market outlook, market drivers, product consumption, production demand, revenue generation, sales volume, and many other important aspects linked to the market dynamics.

Along with focus laid on the important factors that have been positively influencing the Stem Cell Artificial Meat market growth over the forecast timeframe, the analysts have also put forth the restraining factors that are anticipated to slow down business growth in the upcoming future. This is because we believe in complete transparency rather than just shedding praises over the market, so as to sell the report. The challenges anticipated to hamper the market growth are pointed out along with proper guidelines to avoid the unpleasant market situations in the near future.

Key companies operational in the global Stem Cell Artificial Meat market mentioned in the report:

Beyond MeatImpossible FoodsMemphis MeatsBoca FoodsKelloggsMorningstar FarmsSulian

Stem Cell Artificial Meat market By Product Type:

Bovine Stem Cell Artificial MeatFish Stem Cell Artificial MeatOthers

Stem Cell Artificial Meat market By Application Type:

Bovine Stem Cell Artificial MeatFish Stem Cell Artificial MeatOthers

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The key players mentioned above come mapped with their complete strategic profiling correspondingly as where the global scope is considered. Talking about the competitive landscape presented in this report, our research analysts have ensured studying all core capabilities of the industry players. Overall the study helps recognizing the profit-making opportunities and also assists in the design of future plans.

Stem Cell Artificial Meat Market Segments:The report has been segregated into several different segmentscomprising, product type, application, end users, regions, and many other applicable to the Stem Cell Artificial Meat market landscape. Each and every entries belonging to the segments are thoroughly discussed in a simplified structure for better understanding of the market from the readers perspective. Segmental analysis includes breaking down of the individual segments into Industry share, which also includes growth analysis depicted in terms of CAGR scrutinized in the report.

Regional Analysis:Besidessegmental breakdown, the report is highly structured into region wise study. The regional analysis comprehensively done by the researchershighlights key regions and their dominating countries accounting for substantial revenue share in the Stem Cell Artificial Meat market. The study helps understanding how the market will fare in the respective region, while also mentioning the emerging regions growing with a significant CAGR. The following are the regions covered in this report. North America: United States, Canada, and Mexico. South & Central America: Argentina, Chile, and Brazil. Middle East & Africa: Saudi Arabia, UAE, Turkey, Egypt and South Africa. Europe: UK, France, Italy, Germany, Spain, and Russia. Asia-Pacific: India, China, Japan, South Korea, Indonesia, Singapore, and Australia.

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Report Customization:We are always open to report customization. If the downloaded template is not as per your need, please connect with our sales team to initiate the process of customization.Let us know if you have any special requirements focused on a specific segment or region.

Research Methodology:We incorporate both primary and secondary research methodologies to produce highly reliable data and growth assumptions for the future. Our data triangulation method includes analysis of several market scenarios and product mappings, which is then broken down into highly organized and statistical pre-sets.

About Us:Our research base consists of a wide spectrum of premium market research reports. Apart from comprehensive syndicated research reports, our in-house team of research analysts leverages excellent research capabilities to deliver highly customized tailor-made reports. The market entry strategies presented in our reports has helped organizations of all sizes to generate profits by making timely business decisions. The research information including market size, sales, revenue, and competitive analysis offered, is the product of our excellence in the market research domain.

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Stem Cell Artificial Meat : Industry Analysis and Forecast 2020 2025 By Key Players-, Beyond Meat, Impossible Foods, Memphis Meats, Boca Foods -...

Astrocytes Show Protective Role in Early-stage ALS – Technology Networks

Motor neuron disease is a degenerative condition which destroys the nerve cells (motor neurons) in the brain and spinal cord, which control movement, speech, swallowing and breathing. The most common type of motor neuron disease is amyotrophic lateral sclerosis (ALS), which affects around 5,000 people in the UK at any one time.A new study found that in this disease, the motor neurons in the brain and spinal cord become sick and die when a protein, called TDP-43, misfolds and accumulates in the wrong place within the motor neurons. Conversely, when this happens in a type of cell that supports motor neurons, called astrocytes, these cells appear comparatively resistant and survive.

When these two types of cells are close together, the more-resistant astrocytes are able to protect motor neurons from the misfolded protein. This rescue-mechanism helps the motor neurons, which are needed to control muscles, live longer.

The role astrocytes have played in dealing with toxic forms of TDP-43 in motor neurons has not been previously well documented in motor neuron disease. Its exciting that weve now found that they may play an important protective role in the early-stages of this disease, explains Phillip Smethurst, lead author. This has huge therapeutic potential finding ways to harness the protective properties of astrocytes could pave the way to new treatments. This could prolong their rescue function or find a way to mimic their behavior in motor neurons so that they can protect themselves from the toxic protein.

This research also established a new model for studying motor neuron disease. This new method more closely resembles the disease in patients as it uses healthy human stem cells, derived from skin cells, and spinal cord tissue samples donated by patients with motor neuron disease, collected post-mortem.

It is thanks to the selfless donations from people with motor neuron disease, that we were able to study the interplay between motor neurons and astrocytes in conditions that closely resemble what happens in humans. These human cell models are a powerful tool for further studies of motor neuron disease and in the hunt for effective therapies. explains Katie Sidle, co-senior author.

For the first time, we have been able to create a model of sporadic motor neuron disease by essentially transferring the toxic TDP-43 protein from post-mortem tissue into healthy human stem cell-derived motor neurons and astrocytes in order to understand how each cell type responds to this insult, both in isolation and when mixed together. The insights made in this work are testament to the power of creative collaboration and interdisciplinarity. It is through many years working together as a group of clinicians, pathologists, stem cell biologists, protein biochemists and other experts, and with a joint aim of increasing knowledge about motor neuron disease (to ultimately help find a cure), that these results have been possible, says Rickie Patani, co-senior author.ReferenceSmethurst et al. (2020) Distinct responses of neurons and astrocytes to TDP-43 proteinopathy in amyotrophic lateral sclerosis. Brain. DOI: https://doi.org/10.1093/brain/awz419

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Astrocytes Show Protective Role in Early-stage ALS - Technology Networks

Mammals Can Delay The Development of Their Embryos, According to Recent Research – Dual Dove

Recent research sheds light on something quite peculiar, exploring a reproductive mystery that is present in more than 130 species of mammals. A team of researchers conducted by Abdiasis Hussein, an associate director of UW Medicines Institute for Stem Cell, also a UW professor of biochemistry, realized the intriguing findings on mammals.

The results not only bring more details for the understanding of postponed embryo implantation. It also indicates how some quickly splitting cells, such as those present in tumors, turn to be inactive.

To find out what leads to a biochemical hold-and-release on embryonic production, the team provoked diapause in a female mouse by decreasing the estrogen rates. Then, they realized a comparison of the diapause embryos to pre-implantation and post-implantation ones. They also provoked diapause in mouse embryonic stem cells by weakening the cells, and analyze those to actively developing mouse embryonic stem cells.

Researchers had also performed comprehensive investigations of how metabolic and signaling pathways manage both the inactive and active phases of mouse embryos and mouse embryonic stem cells in lab vessels.

Metabolism involves the life-supporting chemical actions cells take out to turn substances into energy, develop materials, and discharge waste. By examining those reactions final actions, dubbed metabolites, the researchers could start to realize the full picture of that occurs to cause diapause and how cells are delivered from its grips.

Bears, seals, weasel-like animals, or armadillos, experience seasonal diapause, as a regular part of their reproductive periods. Many classes of bears, for example, breed in the early stages of spring and sometimes even in early summer. The female then uncontrollable hunts for food, and only when it reaches sufficient weight and body fat, one or more of her embryos implant a few months later after she moves to her cave. Any baby bears would be born in late winter.

Ethelene is the main editor on DualDove, she likes to write on the latest science news.

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Mammals Can Delay The Development of Their Embryos, According to Recent Research - Dual Dove

Banff resident leaves $600,000 legacy to fight blindness – The Crag and Canyon

Doreen Powles legacy will impact people around the world as research discoveries transform lives by leading to treatments that restore sight. Thank you, Doreen, for your generosity, said Doug Earle, President and CEO, Fighting Blindness Canada.

Fighting Blindness Canada said they are grateful for the legacy gift of $600,000 by Banff resident, the late Doreen Marjorie Powles to fight blindness.

The announcement was made in Banff at the annual meeting of the Canadian Retina Society where Canadas retina specialists have gathered to share the latest in research and new treatments.

Fighting Blindness Canada is honoured to announce that Dr. Elizabeth M Simpson at the University of British Columbia is the recipient of the Doreen Powles Award to End Blindness for her project entitled Using gene therapy to Treat Congenital Blindness.

Doreen Powles spent her life in Banff where she lived with a blinding eye disease called congenital blindness. Her blindness was present at birth with reduced ability to detect light and colour, severe nearsightedness and involuntary movements of the eye.

Robert Smyth, executor of Doreen Powles estate said, Doreen was an only child. She left her entire estate to finance eye research, providing scholarships for aspiring researchers and helping people living with blindness. She would be excited by Fighting Blindness Canadas ground-breaking research to restore sight.

G.P. Powles, her father, was a partner in Hornibrook and Powles Insurance in Banff and a Rotarian.

Mr. Powles searched the world at great expense to find a cure for his daughters blindness without success.

Doreen Marjorie Powles graduated in 1952 from the University of British Columbia with a Bachelor of Arts.

Fighting Blindness Canada (FBC) is Canadas leading private funder of vision research. Over our 45-year history, we have invested over $40 million to support vision research and education across Canada with over 200 research grants that have led to over 600 new discoveries in areas such as stem cell research, neuroprotective therapies, technological developments, pharmaceuticals, and gene therapies.

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Banff resident leaves $600,000 legacy to fight blindness - The Crag and Canyon

Exopharm publishes ‘positive and unique’ results for its exosome platform – Small Caps

Regenerative medicine company Exopharm (ASX: EX1) has received amiable results from a BioMAP testing program operated by Eurofins with respect to its exosome products.

Results showed that Exopharms exosome platform is safe in its mechanism of action and that has different and distinct activities compared to 4,500 other drugs.

Strong lab results raise the chances of Exopharms world-leading exosome products of becoming fully-fledged medicines although further testing is required.

The news is expected to have a positive impact across Exopharms business and will be of interest to potential customers, according to the companys chief executive officer Dr Ian Dixon.

The company said the results confirm this therapeutic approach is a distinct and potentially new class of medicine.

However, Exopharm did temper its positivity by admitting that BioMAP testing results may not translate to future testing in non-clinical or clinical trials and said that unforeseen product safety issues may arise at later stages of testing.

Eurofins BioMAP phenotypic profiling and screening service provides an objective, target agnostic and data-driven approach to understanding a medicines impact on human disease models and translational biomarkers.

Validated with clinically approved drugs and known test agents, the BioMAP platform is powered by human primary cell-based disease systems, a reference database of more than 4,500 compounds, data analytics, and expert interpretation to provide clients with actionable insights.

Headline BioMAP results indicated that Exopharms Plexaris product was safe in both relative and absolute terms and had notable biological activity in tissue remodelling, immunomodulatory and inflammatory-related activities.

Exosomes are natural particles produced by cells, delivering whats known as therapeutic cargoes to other cells to reduce inflammation and promote regeneration.

According to researchers, exosomes are plentiful in young individuals but decline with age.

Recent research has indicated that exosomes can be used as a way of extending the number of healthy functional years extending life quality and span.

Moreover, exosomes secreted by stem cells could be used as a substitute for stem-cell therapy with equal or greater benefit while avoiding various problems associated with stem-cell therapy.

Exopharm said exosome could even be used to deliver targeted novel drugs and serve a diagnostic function within various applications.

In parallel to Plexaris, Exopharms Cevaris product was compared with 4,500 experimental and sold medicines across a panel of 12 human primary cell-based systems.

Cevaris was found to be safe and had notable biological activity in the same areas as Plexaris.

Importantly, neither was shown to be cytotoxic, and neither caused antiproliferative effects at the concentrations tested.

Another key finding was that comparing the screening profiles of both Plexaris and Cevaris against the database of 4,500 medicines did not produce a significant match, thereby suggesting that both Plexaris and Cevaris have different and distinct activities in comparison to existing drugs.

This is a valuable finding, pointing to exosomes as a unique and potentially new class of medicine, with potential application unmet by existing medicines, the company said.

These are very positive results from a detailed external test of two of our experimental exosome products, said Dr Ian Dixon.

The testing showed that both Plexaris and Cevaris had different and distinct activities to comparison drugs. This confirms our belief that exosomes are a distinct and potentially new class of medicine, different from existing medicines.

The results of the BioMAP testing will help Exopharm plan its next studies with additional insights and confidence. After that, further human clinical trials are the next step, said Dr Dixon.

Moving forward, Exopharm said the results indicate several potential mechanisms of action and biological pathways for Plexaris and Cevaris, that will be verified in planned upcoming non-clinical studies.

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Exopharm publishes 'positive and unique' results for its exosome platform - Small Caps

Medical Aesthetics Market Is Expected To Grow At CAGR Of 11.5% From 2019 To Reach $22.2 Billion By 2 (1) – PharmiWeb.com

Meticulous Research leading global market research company published a research report titled Medical Aesthetics Market by Product (Facial Aesthetics, Cosmetic Implants, Skin Aesthetic Devices, Thread Lift Products, Body Contouring Devices, Hair Removal Devices), End User (Hospitals, Medical Spas, Home Care Settings)Global Forecast to 2025.

According to this latest publication from Meticulous Research, the global medical aesthetics market is expected to grow at a CAGR of 11.5% from 2019 to reach $22.2 billion by 2025. The growth of this market will be driven by factors such as increasing adoption of minimally invasive and noninvasive aesthetic procedures, increasing public awareness about cosmetic procedures, and rising adoption of medical aesthetic procedures among geriatric population to improve their appearance. However, risk and complications associated with medical aesthetic procedures may hamper the growth of the market to a certain extent.

Get Inside Scoop Of The Report, Download For Free Sample PDF @https://www.meticulousresearch.com/download-sample-report/cp_id=5028(Note: Sample PDF Download with TOC, Charts, and Graphs)

Key questions answered in the report-Which are the high growth market segments in terms of products, technology, disease type, end user, and region/countries?What was the historical market for medical aesthetics market across the globe?What are the market forecasts and estimates for the period 2018-2025?What are the major drivers, opportunities, and challenges in the global medical aesthetics market?Who are the major players in the global medical aesthetics market?How is the competitive landscape and who are the market leaders in the global medical aesthetics market?What are the recent developments in the global medical aestheticsmarket?What are the different strategies adopted by the major players in the global medical aesthetics market?What are the geographical trends and high growth regions/ countries?Who are the local emerging players in the global medical aesthetics market and how do they compete with the global players?

Have Any Query? Ask Our Experts Here:https://www.meticulousresearch.com/speak-to-analyst/cp_id=5028

The global medical aesthetics market study presents historical market data in terms of values (2017 and 2018), estimated current data (2019), and forecasts for 2025- by product (facial aesthetics, cosmetic implants, skin aesthetic devices, physician-dispensed cosmeceuticals and skin lighteners, thread lift products, body contouring devices, hair removal devices, tattoo removal devices, and nail treatment laser devices), and end user (hospitals, clinics, and medical spas, beauty centers, and home care settings). The study also evaluates industry competitors and analyzes the market at a regional and country level.

On the basis of product type, facial aesthetics market segment is estimated to command the largest share of the global medical aesthetics market in 2019; whereas, cosmetic implants segment is expected to grow at the highest CAGR during the forecast period due to increasing growth of minimally invasive reconstruction surgeries, technological advancements such as injectable fillers and gummy bear breast implants, rising number of congenital face disorders, and increasing awareness about aesthetic appearance. A comprehensive range of cosmetic plastic surgery techniques and implants have been developed over the years for improving the appearance or restoring function to the human body. The implants used in cosmetic procedures are to enhance the aesthetic looks of an individual and rectify the deformities caused due to accidents, trauma, and congenital disorders.

On the basis of end user, hospitals, clinics, and medical spas segment is estimated to hold the largest share of the global medical aesthetics market in 2019. Hospitals, clinics, and medical spas are typically well-equipped with technologically advanced instruments/devices and have skilled professionals to provide effective cosmetic treatment to its patients. This has led to their greater share in the global medical aesthetics market. Hospital and medical spas design their services to improve the aesthetic procedures for patient well-being. Medical aesthetic services involve highly advanced technology that needs the combination of healthcare and beauty services. In aesthetic treatment, advanced technologies are increasingly being used to provide medical procedures designed to offer significant cosmetic change for patients. The growth of the hospitals, clinics, and medical spas segment in the market is primarily driven by the growing number of patients undergoing cosmetic procedures to enhance self-esteem, increasing healthcare expenditure on cosmetic procedures, growing geriatric population, and greater uptake of technologically advanced medical aesthetics devices in the hospitals and clinics to provide effective treatments to the patients.

Browse key industry insights spread across 195 pages with 185 market data tables & 18 figures & charts from the market research report:https://www.meticulousresearch.com/product/medical-aesthetics-market-5028/

Geograhic Review:

This research report analyzes major geographies and provides comprehensive analysis of North America (U.S., Canada), Europe (Germany, France, Italy, Spain, and U.K.), Asia-Pacific (China, India, South Korea, Japan, and Australia), Latin America, and Middle East & Africa. North America commanded the largest share of the global medical aesthetics market, followed by Europe and Asia Pacific. The largest share of North American region in the medical aesthetics market is primarily attributed to the growing healthcare sector, increasing awareness and adoption of aesthetic procedures among the population, growing healthcare expenditure, rising incidences of skin diseases, growing geriatric population, various technological advancements, and increase in the consciousness about physical appearances.

Asia Pacific region is expected to be the fastest growing geographic markets for medical aesthetics devices with countries, such as China, Japan, South Korea and India among others for being the largest contributors to the growth of the market. Additionally, rapid urbanization, increasing investments by healthcare providers towards infrastructure improvement, increasing awareness among the population, growing beauty consciousness, and availability of increasing range of advanced products and technologies is contributing to the growth of the medical aesthetics market in the Asia Pacific region.

Access Free Complete Free Sample PDF Copy Here:https://www.meticulousresearch.com/request-sample-report/cp_id=5028

Key players:

The major players operating in the globalmedical aesthetics marketare Allergan plc (Ireland), Alma Lasers (Israel), Anika Therapeutics, Inc. (US), Cutera, Inc. (US), Cynosure Inc. (US), El.En. S.P.A. (Italy), Fotona D.O.O (Solvenia& US), Galderma Laboratories, L.P. (US), Mentor Worldwide LLC (US), and Merz Aesthetics (Germany) among others.

Related Report:

Surgical Sutures Market by Product (Suture Thread (Synthetic, Nylon, Silk, Prolene, Steel), Automatic Suture Device), Application (CVD, General, Orthopedic, Gynec, Ophthalmic, Plastic, Cosmetics), End User (Hospitals, ASC, Clinic) Global Forecast to 2024

Flow Cytometry Market by Product (Cell Analyzers, Cell Sorter, Software, Reagents), Technology (Cell Based, Bead Based), Application (Drug Discovery, Stem Cell Research, Cancer, Organ Transplant, Commercial) and by End-user Global Forecast to 2027

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Medical Aesthetics Market Is Expected To Grow At CAGR Of 11.5% From 2019 To Reach $22.2 Billion By 2 (1) - PharmiWeb.com

(2020-2026) Latest Report shows drastic growth for Stem Cell Therapy Market: Which factor will limit market growth?| Osiris Therapeutics, NuVasive,…

Research report on global Stem Cell Therapy market 2020 with industry primary research, secondary research, product research, size, trends and Forecast.

The report offers highly detailed competitive analysis of the Global Stem Cell Therapy industry, where the business and industry growth of leading companies are thoroughly evaluated on the basis of production, product portfolio, recent developments, technology, geographical footprint, and various other factors. The authors of the report have also provided information on future changes in the competitive landscape and the expected nature of competition in the global Stem Cell Therapy industry. This will help players to prepare themselves well for any unforeseen situations in the industry competition and give a tough competition to other players in the global Stem Cell Therapy industry.

Click here! For Updated Sample Copy of this [emailprotected]: https://www.qyresearch.com/sample-form/form/1436410/global-Stem-Cell-Therapy-market

As part of geographic analysis of the global Stem Cell Therapy market, the report digs deep into the growth of key regions and countries, including but not limited to North America, the US, Europe, the UK, Germany, France, Asia Pacific, China, and the MEA. All of the geographies are comprehensively studied on the basis of share, consumption, production, future growth potential, CAGR, and many other parameters.

The following players are covered in this report:Osiris TherapeuticsNuVasiveChiesi PharmaceuticalsJCR PharmaceuticalPharmicellMedi-postAnterogenMolmedTakeda (TiGenix)Stem Cell Therapy Breakdown Data by TypeAutologousAllogeneicStem Cell Therapy Breakdown Data by ApplicationMusculoskeletal DisorderWounds & InjuriesCorneaCardiovascular DiseasesOthers

Regions Covered in the Global Stem Cell Therapy Market:

The Middle East and Africa (GCC Countries and Egypt) North America (the United States, Mexico, and Canada) South America (Brazil etc.) Europe (Turkey, Germany, Russia UK, Italy, France, etc.) Asia-Pacific (Vietnam, China, Malaysia, Japan, Philippines, Korea, Thailand, India, Indonesia, and Australia)

Highlights of the Report Accurate market size and CAGR forecasts for the period 2020-2025 Identification and in-depth assessment of growth opportunities in key segments and regions Detailed company profiling of top players of the global Stem Cell Therapy market Exhaustive research on innovation and other trends of the global Stem Cell Therapy market Reliable industry value chain and supply chain analysis Comprehensive analysis of important growth drivers, restraints, challenges, and growth prospects

What the Report has in Store for you?

Get Customized Report in your Inbox within 24 hours @https://www.qyresearch.com/customize-request/form/1436410/global-Stem-Cell-Therapy-market

Table of Contents

Report Overview:It includes six chapters, viz. research scope, major manufacturers covered, market segments by type, Stem Cell Therapy market segments by application, study objectives, and years considered.

Global Growth Trends:There are three chapters included in this section, i.e. industry trends, the growth rate of key producers, and production analysis.

Stem Cell Therapy Market Share by Manufacturer:Here, production, revenue, and price analysis by the manufacturer are included along with other chapters such as expansion plans and merger and acquisition, products offered by key manufacturers, and areas served and headquarters distribution.

Market Size by Type:It includes analysis of price, production value market share, and production market share by type.

Market Size by Application:This section includes Stem Cell Therapy market consumption analysis by application.

Profiles of Manufacturers:Here, leading players of the global Stem Cell Therapy market are studied based on sales area, key products, gross margin, revenue, price, and production.

Stem Cell Therapy Market Value Chain and Sales Channel Analysis:It includes customer, distributor, Stem Cell Therapy market value chain, and sales channel analysis.

Market Forecast Production Side: In this part of the report, the authors have focused on production and production value forecast, key producers forecast, and production and production value forecast by type.

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QYResearch always pursuits high product quality with the belief that quality is the soul of business. Through years of effort and supports from the huge number of customer supports, QYResearch consulting group has accumulated creative design methods on many high-quality markets investigation and research team with rich experience. Today, QYResearch has become a brand of quality assurance in the consulting industry.

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(2020-2026) Latest Report shows drastic growth for Stem Cell Therapy Market: Which factor will limit market growth?| Osiris Therapeutics, NuVasive,...

2nd Annual Cell Therapy Bioprocessing Conference | Boston, MA, USA – June 25-26, 2020 | Network with Key Professionals Addressing the Strategies of…

Dublin, Feb. 12, 2020 (GLOBE NEWSWIRE) -- The "2nd Annual Cell Therapy Bioprocessing Conference" conference has been added to ResearchAndMarkets.com's offering.

Over the last decade, the field of cell therapy has rapidly grown, and it holds enormous promise for treating many diseases. In the conference of 2017, specific risks and benefits were assessed of the cell culture and cell therapy bio-manufacturing for the cure of these diseases.

There are still factors like manufacturing maze, investment, logistics and regulatory challenges which prevents the cell and gene therapies to be widely used.

2nd Annual Cell Therapy Bioprocessing Conference will put together a unique platform to provide the exact solutions to these robust manufacturing and bioprocessing challenges, taking place in Boston-USA on 25th & 26th June 2020.

Key Highlights

Why Attend?

Agenda

Day 1: Thursday June 25th

CELL CULTURE TO CELL THERAPY

Self-regulated bioprocessing through bioreactor system for monitored 3D cell culture09:00 - 09:30

Multi-omics study on CHO cell lines engineering and bioprocessing09:30 - 10:00

Solution provider presentation10:00 - 10:30

Morning refreshment and One on One Networking10:30 - 11:20

CELL THERAPY BIOPROCESSING AND DEVELOPMENT

Next generation engineered T cells for cell therapy11:20 - 11:50 Jan Joseph Melenhorst, Adjunct Associate Professor, Upenn

Automation of cellular therapies: challenges and solutions11:50 - 12:20

Solution provider presentation12:20 - 12:50

Lunch and One on One Networking12:50 - 13:50

Development of Stem Cell Derived Exosomes for Clinical Applications13:50 - 14:20 Ian McNiece, Vice President, Aegle Therapeutics

Bioprocessing of recombinant adenoassociated virus production by fluorescence spectroscopy14:20 - 14:50

Afternoon refreshment and One on One Networking14:50 - 15:20

PROCESS MONITORING & QUALITY CONTROL

Process development of antigen-specific T cells16:10 - 16:40 Patrick J. Hanley, Director of GMP for Immunotherapy, The Children's Research Institute

Establishing iPSC cell banks derived using reagents and workflows optimized for cell therapy manufacturing16:40 - 17:10

Glycolysis restriction through fermentation technology to preserve T-cell functions and checkpoint therapy17:10 - 17:40

Closing remarks by Chairperson17:40 - 17:45

Drinks reception17:45 - 18:45

End of Conference18:45 - 18:45

Day 2: Friday June 26th

Scale up of allogeneic cell therapy manufacturing in single-use bioreactors: Challenges, insights and solutions09:00 - 09:30

Cell therapy: progress in manufacturing and assessments of potency09:30 - 10:00

Solution provider presentation10:00 - 10:30

Morning refreshment and One on One Networking10:30 - 11:20

Panel Discussion: Autologous vs Allogenic11:20 - 11:50

Quality control and effective purification methods for cell therapy product lines11:50 - 12:20

Solution provider presentation12:20 - 12:50

Lunch and One on One Networking12:50 - 13:50

LOGISTICS, REGULATORY & INVESTMENT

Building a leading edge supply chain, while maintaining flexibility13:50 - 14:20 Devyn Smith, Chief Operating Officer, Sigilon Therapeutics

Raw and starting material control for cell-based medicines14:20 - 14:50

FDA guidelines for regulatory issues involved cell therapy manufacturing14:50 - 15:20

Closing remarks by Chairperson15:20 - 15:25

End of Conference15:25 - 15:25

For more information about this conference visit https://www.researchandmarkets.com/r/jhs3n8

Research and Markets also offers Custom Research services providing focused, comprehensive and tailored research.

CONTACT: ResearchAndMarkets.comLaura Wood, Senior Press Managerpress@researchandmarkets.comFor E.S.T Office Hours Call 1-917-300-0470For U.S./CAN Toll Free Call 1-800-526-8630For GMT Office Hours Call +353-1-416-8900

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2nd Annual Cell Therapy Bioprocessing Conference | Boston, MA, USA - June 25-26, 2020 | Network with Key Professionals Addressing the Strategies of...

CRISPR Therapeutics Provides Business Update and Reports Fourth Quarter and Full Year 2019 Financial Results – Yahoo Finance

-Enrollment ongoing in clinical trials of CTX001 for patients with severe hemoglobinopathies-

-Enrollment ongoing in clinical trial of CTX110, targeting CD19+ malignancies-

-Enrollment has begun in clinical trial of CTX120, targeting B-cell maturation antigen (BCMA)-

ZUG, Switzerland and CAMBRIDGE, Mass., Feb. 12, 2020 (GLOBE NEWSWIRE) -- CRISPR Therapeutics(CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today reported financial results for the fourth quarter and full year ended December 31, 2019.

In 2019, CRISPR Therapeutics achieved important milestones and momentum across key programs. We announced positive interim safety and efficacy data from the first two patients in our ongoing CTX001 clinical trials, one patient with beta thalassemia and one patient with sickle cell disease. These preliminary data support our belief in the potential of CTX001 to have meaningful benefit for patients following a one-time intervention, said Samarth Kulkarni, Ph.D., Chief Executive Officer of CRISPR Therapeutics. In addition, we advanced our first allogeneic CAR-T cell therapy, CTX110, targeting CD19+ malignancies and, building on this progress, today announced that we have begun enrolling patients in a clinical trial for our second allogeneic CAR-T therapy, CTX120, targeting BCMA for the treatment of relapsed or refractory multiple myeloma.

Dr. Kulkarni added: 2020 has the potential to be a pivotal year in our companys growth. We expect to conduct clinical trials in five indications, and we anticipate new data from our immuno-oncology and hemoglobinopathies programs. Our continued progress brings us closer to potentially providing transformative therapies to patients with serious diseases.

About CTX001TMCTX001 is an investigational ex vivo CRISPR gene-edited therapy that is being evaluated for patients suffering from TDT or severe SCD in which a patients hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin (HbF; hemoglobin F) in red blood cells. HbF is a form of the oxygen-carrying hemoglobin that is naturally present at birth and is then replaced by the adult form of hemoglobin. The elevation of HbF by CTX001 has the potential to alleviate transfusion requirements for TDT patients and painful and debilitating sickle crises for SCD patients.

CTX001 is being developed under a co-development and co-commercialization agreement between CRISPR Therapeutics and Vertex.

About CTX110TMCTX110 is a healthy donor-derived gene-edited allogeneic CAR-T therapy targeting cluster of differentiation 19, or CD19, for the treatment of CD19+ malignancies. A wholly-owned asset of CRISPR Therapeutics, CTX110 is in a clinical trial designed to assess the safety and efficacy of CTX110 in relapsed or refractory B-cell malignancies. The multi-center, open-label clinical trial is designed to enroll up to 95 patients and investigate several dose levels of CTX110.

About CTX120TMCTX120 is a healthy donor-derived gene-edited allogeneic CAR-T therapy targeting B-cell maturation antigen, or BCMA. A wholly-owned asset of CRISPR Therapeutics, CTX120 is in a clinical trial designed to assess the safety and efficacy of CTX120 in relapsed or refractory multiple myeloma. The multi-center, open-label clinical trial is designed to enroll up to 80 patients and investigate several dose levels of CTX120.

About CTX130TMCTX130 is a healthy donor-derived gene-edited allogeneic CAR-T therapy targeting CD70, an antigen expressed on hematologic cancers. A wholly-owned asset of CRISPR Therapeutics, CTX130 is in development for the treatment of both solid tumors, such as renal cell carcinoma, and T-cell and B-cell hematologic malignancies.

About CRISPR TherapeuticsCRISPR Therapeutics is a leading gene editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer, Vertex Pharmaceuticals and ViaCyte, Inc. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Cambridge, Massachusetts, and business offices in San Francisco, California and London, United Kingdom. For more information, please visit http://www.crisprtx.com.

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Editing a -globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype – Science Advances

INTRODUCTION

-Hemoglobinopathies (SCD and -thalassemia) are severe anemias characterized by abnormal or reduced production of hemoglobin (Hb) chains. SCD and -thalassemia are the most common monogenic disorders with an incidence of 1 per 318,000 live births worldwide. In -thalassemia, the reduced production of chains causes -globin precipitation and insufficiently hemoglobinized red blood cells (RBCs). In SCD, the 6GluVal substitution leads to Hb polymerization and RBC sickling, which is responsible for vaso-occlusive crises, hemolytic anemia, and organ damage.

Allogeneic hematopoietic stem cell (HSC) transplantation is the only definitive cure for patients affected by SCD or -thalassemia. Transplantation of autologous, genetically modified HSCs represents a promising therapeutic option for patient lacking a compatible HSC donor (1). Pioneering clinical trials based on lentiviral (LV)based gene addition approaches demonstrated a clinical benefit in -thalassemic patients with residual -globin production (+-thalassemia). However, this treatment is, at best, partially effective in correcting the clinical phenotype of severe 0-thalassemia (no residual -globin production) and SCD patients, where higher levels of therapeutic globin are required to restore correct globin chain balance and inhibit HbS polymerization (26).

The clinical severity of -hemoglobinopathies is alleviated by the co-inheritance of genetic mutations causing a sustained fetal -globin chain production at adult age, a condition termed hereditary persistence of fetal Hb (HPFH) (7). Elevated fetal -globin levels reduce globin chain imbalance in -thalassemias and exert a potent anti-sickling effect in SCD. Compared with current LV-based gene addition approaches, therapeutic strategies aimed at forcing a -globinto-globin switch (8) have the advantage of guaranteeing high-level expression of the endogenous -globin genes and, in the case of SCD, reduction of the S-globin synthesis.

HPFH mutations and single-nucleotide polymorphisms (SNPs) associated with HbF levels of up to 40% of the total Hb were identified at positions 200, 175, 158, and 115 upstream of the HBG1 and HBG2 transcription start sites (TSSs) (Fig. 1A). These mutations either generate de novo DNA motifs recognized by transcriptional activators (9, 10) or disrupt the binding sites for transcriptional repressors. In particular, HPFH mutations in the 200 and 115 regions reduce the binding of LRF and BCL11A transcriptional repressors, respectively, thus inhibiting -globin silencing (11, 12). In addition, SNPs at position 158 of both HBG -globin promoters are associated with enhanced -globin expression (1317). These SNPs might either identify a putative transcriptional repressor binding site or create a binding site for a transcriptional activator. An ideal and universal strategy to correct the clinical phenotype of patients with -hemoglobinopathies would be to introduce HPFH mutations in the -globin promoters via homology-directed repair (11), which, however, is inefficient in HSCs (18). Here, we mimicked HPFH mutations by disrupting known or putative binding sites for transcriptional repressors in the -globin promoters using a CRISPR-Cas9based genome editing strategy that takes advantage of the nonhomologous end joining (NHEJ) and microhomology (MH)mediated end joining (MMEJ)mediated DNA repair mechanisms to induce insertions and deletions (InDels) within the -globin repressor DNA binding motifs. In particular, we show that efficient disruption of known (200) or putative (158) binding sites via CRISPR-Cas9 leads to HbF derepression and thus mimics the effect of HPFH mutations and SNPs in erythroid cell lines and in RBCs derived from SCD patients hematopoietic stem/progenitor cells (HSPCs). Targeting the LRF-binding site corrects the SCD cell phenotype and is effective in repopulating HSPCs.

(A) Schematic representation of the -globin locus on chromosome 11, depicting the hypersensitive sites of the locus control region (white boxes) and the HBE1, HBG2, HBG1, HBD, and HBB genes (colored boxes). The sequence of the HBG2 and HBG1 identical promoters (from 210 to 100 nucleotides upstream of the HBG TSS) is shown below. Black arrows indicate HPFH mutations described at HBG1 and/or HBG2 promoters, with the percentage of HbF in heterozygous carriers of HPFH mutations (42). The highest HbF levels were generally observed in individuals carrying SCD (*) or -thalassemia mutations (**). LRF- and BCL11A-binding sites [as described in (11)] are highlighted by orange and green boxes, respectively. The 114/102 13-bp HPFH deletion is indicated by an empty box. Red arrows indicate the gRNA cleavage sites. (B to E) Globin expression analyses were performed in mature erythroblasts differentiated from Cas9-GFP+ HUDEP-2 cells. Results are shown as means SEM of three to four independent experiments. (B) RT-qPCR quantification of (G + A)- and -globin transcripts. mRNA levels were expressed as percentage of ( + ) globins, after normalization to -globin mRNA levels. (C) Representative flow cytometry plots showing the percentage of HbF+ cells. (D) RP-HPLC analysis of globin chains. -Like globin expression was normalized to -globin. Representative RP-HPLC chromatograms are reported together with the expression of -globin chains (in brackets). The ratio of chains to non chains was similar between HBG-edited and control samples. (E) ChIP-qPCR analysis of H3K27Ac at HBB and HBG promoters in 197-edited HUDEP-2 cells and control AAVS1edited samples (day 5 of differentiation, n = 3). ChIP was performed using an antibody against H3K27Ac and the corresponding control immunoglobulin G (IgG). ****P 0.0001, ***P 0.001, **P 0.01, and *P 0.05 (unpaired t test). SSC, side scatter. (F) ChIP-qPCR analysis of LRF at HBG promoters in 197-edited and control AAVS1edited K562 cells (n = 2 biologically independent experiments). ChIP was performed using an antibody against LRF. Two different primer pairs were used to amplify the HBG promoters (A and B). KLF1 and DEFB122 served as positive and negative controls, respectively.

We designed guide RNAs (gRNAs) targeting the 200 LRF-binding site (197, 196, and 195) and the 158 region (158, 152, and 151) (Fig. 1A). In parallel, we used a gRNA targeting the 115 region (115) that was reported to induce HbF reactivation by generating a 13base pair (bp) deletion spanning the BCL11A-binding site (19) and a control gRNA targeting the unrelated AAVS1 locus. Plasmid delivery of individual gRNAs and a Cas9green fluorescent protein (GFP) fusion in the erythroleukemia cell line K562 revealed a similar editing efficiency for the gRNAs targeting the 200 region, whereas the 158 gRNA showed the highest editing efficiency at the 158 region. High cleavage efficiency was also observed for the 115 and AAVS1 gRNAs (fig. S1A).

We next used the HUDEP-2 adult erythroid cell line to evaluate -globin derepression following disruption of the 200, 158, and 115 regions. After plasmid transfection, bulk populations of Cas9-GFP+ HUDEP-2 cells were differentiated into mature erythroblasts. Overall, genome editing efficiency was ~80% for all the gRNAs tested, with the exception of the 158 gRNA (50 4%; fig. S1B). The editing frequency was similar at days 0 and 9 of erythroid differentiation, thus showing that edited cells were not counterselected during erythroid maturation (fig. S1B). The presence of a 158 C>T heterozygous SNP in the HBG2 promoter resulted in a reduced editing of HBG2 compared to HBG1 with the gRNA 158 (40 6% versus 68 1%; fig. S1C). Similar editing frequencies at the HBG1 and HBG2 promoters were observed with the other gRNAs (fig. S1C). Deep sequencing analysis revealed that virtually all the editing events altered the LRF- and BCL11A-binding sites in 200 and 115 edited samples, respectively, mostly through small deletions (fig. S1D). In a fraction of -globin loci, simultaneous cleavage of the HBG promoters resulted in the deletion of the intervening 4.9-kb genomic region and loss of the HBG2 gene, with a frequency ranging from 9 1% to 16 3% (fig. S1E).

Editing of the HBG promoters did not alter erythroid cell differentiation, as assessed by morphological analysis, and flow cytometry and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of erythroid markers (fig. S2, A to C). Disruption of the 200 region increased the production of -globin transcripts and a parallel decrease of adult -globin and -globin mRNA synthesis (Fig. 1B and fig. S2D). Similar changes were observed upon targeting the 115 region, while a lower -globin reactivation was observed upon targeting the 158 region (Fig. 1B and fig. S2D). -Globin mRNA levels were not significantly different among HBG-edited and control samples (fig. S2D). Flow cytometric analysis of cells edited at 197, 196, and 195 positions revealed a high frequency of HbF-expressing cells (F cells) (79 1%, 71 1%, and 78 1%). Similar results were obtained by disrupting the 115 region (71 3%), while a lower percentage of F cells (43 5%) was obtained in the 158 edited samples (Fig. 1C). Reversed-phase high-performance liquid chromatography (RP-HPLC) confirmed the significant increase in -globin with concomitant decrease of -globin production following editing of the 200 and the 115 regions, while 158 edited cells displayed a milder increase in -globin levels (Fig. 1D). Targeting the LRF-binding site resulted in high HbF levels, accounting for up to 28 1% of the total Hb in 197 samples, as determined by cation-exchange HPLC (CE-HPLC). Cells edited with the 115 gRNA showed comparable HbF reactivation (24 3%), while 158 edited cells showed HbF levels of only 5 2% (fig. S2E). HbF mainly contained A (HBG1) rather than G (HBG2) chains, which could be explained by loss of HBG2 caused by the 4.9-kb deletion (fig. S2F). Moreover, cells carrying the 4.9-kb deletion may reactivate more potently -globin expression, as the HBG1-HBG2 intervening sequence might contain cis regulatory elements that repress HBG transcription. HBG-edited HUDEP-2 showed a normal chain/non chain ratio, indicating that the increased production of -globin chains compensated for the reduction of -globin synthesis (Fig. 1D).

Disruption of the LRF-binding site at both HBG promoters was associated with increased H3K27 acetylation (H3K27Ac), a marker of active regulatory elements (Fig. 1E). Concomitantly, H3K27Ac tended to be reduced at the HBB gene in 197 edited cells compared to control samples (Fig. 1E). As LRF binding cannot be detected at the HBG promoters in wild-type HUDEP-2 cells expressing low HbF levels (11), we evaluated LRF binding in HbF+ K562 edited using the 197 gRNA (66% of editing efficiency) or the AAVS1 control gRNA (72% of editing efficiency). Chromatin immunoprecipitation (ChIP)qPCR experiments showed a twofold reduction in LRF binding in 200-edited cells.

To test the anti-sickling properties of induced -globin synthesis in a clinically relevant model, we edited the -globin repressor binding sites in CD34+ HSPCs obtained from SCD patients by plerixafor mobilization (20). We first optimized a selection-free, ribonucleoprotein (RNP)based protocol (21) to efficiently edit the HBG promoters in CD34+ HSPCs. The use of chemically modified single gRNAs in combination with a transfection enhancer oligonucleotide resulted in the editing of up to 75% of the alleles using the gRNAs targeting the 200 region (fig. S3A). SCD HSPCs were then transfected with RNP complexes containing either the gRNAs targeting the HBG promoters or the control AAVS1 gRNA. Following erythroid differentiation, genome editing efficiency in bulk populations of mature erythroblasts achieved values of 80% in cells transfected with 197, 196, 195, and 115 gRNAs (Fig. 2A and fig. S3B). Editing frequency with the 158 gRNA was variable because of the presence of the C>T SNP at that position in a fraction of the SCD donors (Fig. 2A and fig. S3B). Genome editing efficiency was similar between the HBG2 and HBG1 promoters, except for samples harboring the 158 SNP and treated with the 158 gRNA (fig. S3B). Of note, the deletion of the 4.9-kb intervening region between HBG2 and HBG1 promoters was not detected in any of the edited primary samples (fig. S3C). This discrepancy between deletion efficiency in HUDEP-2 and HSPCs was also observed in previous studies targeting the 115 region (19, 22) and might be ascribed to a higher expression of the CRISPR-Cas9 system in HUDEP-2 cells [transfected with plasmids and FACS (fluorescence-activated cell sorting)sorted on the basis of Cas9-GFP expression] that favors the simultaneous cleavage of the HBG promoters (23). However, we cannot exclude that transformed cell lines might be more prone to illegitimate repair and can cope easily with large deletions.

(A) Deep sequencing analysis of genome editing events in mature erythroblasts derived from adult SCD and CB healthy donor HSPCs. The InDel profile was unchanged between SCD and healthy donor cells. Frequencies of substitutions (subst), insertions (ins), and deletions (del) are shown as percentages of total InDels. The proportion of >1-bp deletions associated or not with MH motifs is indicated. The frequency of >1-bp deletions associated with MH motifs was significantly lower for the 196 gRNA compared to the 197 (P 0.01) and 195 (P 0.001) gRNAs. Data are expressed as means SEM (n = 3 to 4, two to three donors). (B) Genome editing efficiency in BFU-E and CFU-GM progenitors derived from edited SCD HSPCs as evaluated by TIDE. Data are expressed as means SEM (n = 2 to 5, two SCD donors). (C) Genome editing in single BFU-E and CFU-GM colonies derived from SCD HSPCs as evaluated by TIDE. We plotted the number of edited HBG promoters. In the 158 sample, the donor did not harbor the 158 SNP. (D) InDel profiles generated by each gRNA as analyzed by deep sequencing. The length of MH motifs associated with specific InDels is indicated. Data are expressed as means SEM (n = 3 to 4, two to three donors). (E) Genome editing efficiency in subpopulations of 197- and 196-edited CB-derived HSPCs. Cells were FACS-sorted based on the expression of CD34, CD133, and CD90, and genome editing efficiency was determined in committed (CD34+CD133), early (CD34+CD133+CD90), and primitive (CD34+CD133+CD90+) progenitors. We plotted the data of three independent experiments starting from unsorted HSPCs with low, medium, and high genome editing efficiency (three donors).

Control and edited SCD HSPCs were plated in clonogenic cultures [colony-forming cell (CFC) assay], allowing the growth of erythroid [burst-forming uniterythroid (BFU-E)] and granulomonocytic [colony-forming unitgranulomonocytic (CFU-GM)] progenitors. Genome editing efficiency was comparable in pools of BFU-Es and CFU-GMs that showed a similar InDel profile (Fig. 2B and fig. S3D). Clonal analysis of single CFCs revealed that >85% of hematopoietic progenitors were edited at the target sites, with ~86 and ~67% of BFU-Es and CFU-GMs, respectively, displaying 3 edited HBG promoters (Fig. 2C). Transfection with the full RNP complex reduced the number of hematopoietic progenitors by 10 to 50% compared to transfection of Cas9 protein alone (fig. S3E).

Previous reports have suggested that HSCs, the target of therapeutic genome editing, are preferentially edited via the NHEJ mechanism (24, 25). On the contrary, MMEJ repair pathway, which takes place through annealing of short stretches of identical sequence flanking the double-strand break (DSB), may be less active (24, 25). Therefore, for each gRNA, we evaluated the frequency of mutations with or without MH motifs as a proxy for the relative contribution of MMEJ- and NHEJ-mediated events. In HSPC-derived erythroid bulk populations, among the editing events, deletions were predominant, and a variable fraction of them (30 to 50%) were associated with the presence of MH motifs in the target sequence (Fig. 2A). In particular, MMEJ events at the LRF-binding site can be likely caused by the presence of two stretches of four cytidines (Fig. 1A and table S1). Among the total InDels, the frequency of events associated with MH motifs was significantly higher for the 197 (38 3%) and 195 (32 1%) gRNAs than for the 196 gRNAs (23 1%). The gRNAs targeting the LRF-binding site induced distinct InDel profiles: 196- and 195-edited cells harbored mainly 1-bp insertions and 1- to 2-bp deletions, while the 197 gRNAs generated the largest fraction of >2-bp deletion events, of which ~45% were associated with MH motifs (Fig. 2D and table S1). Virtually all the editing events generated by the 197, 196, and 195 gRNAs disrupted the LRF-binding site (table S1). Of note, the proportion of nucleotides in the LRF-binding site that were lost as a result of editing was higher in 197 than in 196 and 195 samples (fig. S4). As expected, the 115 gRNA caused disruption of the BCL11A-binding site (19). In these samples, 13-bp deletions partially spanning the BCL11A-binding site were associated with an 8-bp MH motif and likely mediated by MMEJ (fig. S4 and table S1) (19). Last, the 158 gRNA generated mostly 1-bp insertions and small deletions around the cleavage site (Fig. 2D, fig. S4, and table S1). To evaluate CRISPR-Cas9mediated genetic modification of the CD34+ cell fraction containing more primitive HSPCs, HBG promoter editing was assessed in FACS-isolated HSPC subpopulations (26), after transfection of the 197 and 196 gRNAs, associated with high and low frequencies of deletions associated with MH motifs, respectively. Editing frequencies were comparable between primitive CD34+/CD133+/CD90+ and early CD34+/CD133+/CD90 progenitors and between CD34+/CD133 committed progenitors and unsorted CD34+ cells even in the case of a limited genome editing efficiency, with a similar InDel profile across the different CD34+ cell subpopulations (Fig. 2E and fig. S5). It is noteworthy that deletions potentially generated via MMEJ occurred even in the more primitive, HSC-enriched populations (fig. S5).

To evaluate HbF reactivation and correction of the SCD cell phenotype upon HBG promoter editing, bulk populations of SCD HSPCs were terminally differentiated into enucleated RBCs. Editing of the HBG promoters did not affect erythroid differentiation, as evaluated by flow cytometry and RT-qPCR analysis of stage-specific erythroid markers and RBC enucleation and by morphological analysis (fig. S6, A to C). Editing of the 200 region led to increased levels of -globin mRNAs, which accounted for 48 3% of total -like globin transcripts in cells transfected with the 197 gRNA (Fig. 3A). -Globin mRNA levels were not significantly different among HBG-edited and control samples (fig. S6D). The proportion of F cells in cells transfected with the 197, 196, and 195 gRNAs was 81 1%, 74 2%, and 74 2%, respectively (Fig. 3B). Analysis of 197- and 196-edited erythroblasts sorted by cytofluorimetry based on the intensity of HbF expression revealed a positive correlation between InDel frequency and extent of -globin production, indicating that the efficacy of HbF reactivation likely increases when targeting a higher number of HBG promoters per cell (fig. S6, E and F). Editing of the 115 region led to HBG derepression and a proportion of 80 2% of F cells, while -globin reactivation was less pronounced in the 158 samples (55 5% of F cells; Fig. 3, A and B). It is noteworthy that for the 158 gRNA, HBG derepression was still modest in RBCs derived from HSPCs harboring >85% of edited HBG promoters (Fig. 3, A and B), suggesting that the 158 region contains a sequence that modestly contributes to inhibition of -globin expression in adult cells. This is consistent with the mild increase in HbF known to be associated with the 158 SNPs. However, an alternative hypothesis is that these SNPs generate a DNA motif recognized by a still unknown transcriptional activator; thus, the mechanism of action remains unclear. RP-HPLC showed a significant increase in -globin chain expression and a reciprocal reduction in S-globin levels in the RBC progeny of 200- and 115-edited HSPCs, with no evidence of imbalance in the /non globin chain synthesis (Fig. 3C). In 197-edited cells, the increase in -globin chains and the reduction of S-globin levels resulted in an inversion of the / globin ratio. Comparable A- and G-globin levels were detected in most of the samples analyzed, consistent with the absence of 4.9-kb deletions. However, in 115-edited cells, HbF was mainly composed of A-globin (fig. S6G). Unexpectedly, in the 115 samples, the relative frequency of the various editing events was different between HBG1 and HBG2 promoters, with 13-bp deletions occurring more frequently in HBG2 than in HBG1, while HBG1 editing events were mainly smaller deletions (table S2). This difference in the editing of HBG1 and HBG2 was unexpected and does not obviously explain the altered A/G ratio in 115-edited samples. CE-HPLC confirmed that editing of the 200 region produced an Hb profile comparable to asymptomatic heterozygous carriers, with HbF representing up to 47 3% of the total Hb tetramers (197 samples; Fig. 3D). Total Hb levels were comparable between RBCs derived from HBG-edited and control HSPCs (fig. S6H).

(A) (G + A)- and S-globin transcript levels detected by RT-qPCR in primary mature erythroblasts. Values are expressed as percentage of ( + S)-globin mRNAs after normalization to -globin. (B) Representative flow cytometry plots showing the percentage of HbF+ cells in RBC populations derived from control and HBG-edited SCD HSPCs. (C) RP-HPLC quantification of -, S-, and -globin chains. -Like globin expression was normalized to -globin. The ratio of chains to non chains was similar between control and HBG-edited samples. Data are expressed as means SEM. (D) Quantification of total HbF (HbF + AcHbF), HbS, and HbA2 by CE-HPLC. We plotted the percentage of each Hb type over the total Hb tetramers. (E and F) In vitro sickling assay of RBCs derived from edited SCD HSPCs under hypoxic conditions (0% O2). (E) Representative photomicrographs of RBCs derived from control and HBG-edited SCD HSPCs at 0% O2. Scale bar, 20 m. (F) Proportion of non-sickled RBCs (0% O2). (A to F) Data are expressed as means SEM (n = 3 to 7, two SCD donors). ****P 0.0001, ***P 0.001, **P 0.01, and *P 0.05 versus AAVS1 sample (unpaired t test).

To assess the effect of HbF reactivation on the sickling phenotype, we performed an in vitro deoxygenation assay that induces sickling of RBCs under hypoxia. At an oxygen concentration of 0%, ~80% of control SCD RBCs acquired a sickled shape (Fig. 3, E and F). Targeting of the 158 region essentially failed to rescue the SCD phenotype (29 13% of nonsickling RBCs; Fig. 3F). In 115-edited samples, HbF reactivation prevented the sickling of 56 9% of RBCs (Fig. 3F). A marked correction of the SCD phenotype was achieved upon disruption of the LRF-binding site, with 69 6% (196) to 79 7% (197) of cells that maintained a biconcave shape under hypoxia (Fig. 3F). Even gRNAs generating predominantly 1- to 2-bp InDels (195 and 196) induced -globin levels that were sufficient to inhibit sickling in a large fraction of RBCs. These results show that editing of the repressor binding sites in the HBG promoters leads to reactivation of HbF sufficient to revert the sickling phenotypes in erythrocytes differentiated from CD34+ HSPCs derived from SCD patients.

Last, in bulk populations of edited SCD erythroblasts, deep sequencing of top-scoring off-targets identified by GUIDE-seq (27) in 293T cells (fig. S7A) showed low to undetectable off-target activity at most of the sites. An average InDel frequency of ~20% was observed in cells edited with the 196 gRNA within an intergenic site located on chromosome 12 (OT-196.1) (fig. S7B). This site lies ~15-kb away from the nearest gene and does not map to known regulatory elements involved in hematopoiesis.

We next evaluated editing efficiency in repopulating HSPCs. Mobilized healthy donor HSPCs were transfected with 197, 196, 115, or AAVS1 gRNAs. We achieved an average editing efficiency of 77.3 3.7%, 87.4 4.6%, and 89.6 2.8% for the 197, 196, 115 gRNAs, respectively, as measured in in vitro cultured HSPCs, and BFU-E and CFU-GM pools (input cells). Untreated and edited cells were injected into NSG immunodeficient mice, and 16 weeks after transplantation, we analyzed the engraftment of human hematopoietic cells and editing efficiency. The engraftment of control and HBG-edited cells was not statistically different, as analyzed in bone marrow, spleen, and thymus (Fig. 4A), with no skewing toward a particular lineage in any of the samples (fig. S8). Editing efficiency in human cells in the bone marrow and spleen, respectively, was 43.0 9.3% and 33.4 4.0% (197), 60.3 6.1% and 62.0 1.7% (196), and 47.6 4.2% and 58.2 3.1% (115) (Fig. 4B). The 197 gRNA showed a similar InDel profile in the input and in the engrafted human cells, with most of MH motifassociated events occurring at a comparable frequency (Fig. 4C). For the 196 gRNA, events associated to MH motifs were significantly reduced in vivo but were already present at a low frequency in the input populations (Fig. 4C) concordantly with the data obtained in mature erythroblasts in vitro (Fig. 2D). Virtually all editing events disrupt the LRF-binding sites in 197 and 196 samples (Fig. 4C). Last, the frequency of the MH motifassociated 13-bp deletion tended to be lower in the progeny of repopulating HSPCs compared to the input samples, as previously reported (Fig. 4C) (24). Together, these results show that the LRF-binding site can be efficiently targeted in engrafting HSPCs.

(A) Engraftment of human cells in NSG mice transplanted with untreated (UT) and edited mobilized healthy donor CD34+ cells (n = 4 mice for each group) 16 weeks after transplantation. Engraftment is represented as percentage of human CD45+ cells in the total murine and human CD45+ cell population, in bone marrow (BM), spleen, thymus, and blood. Values shown are means SEM; *P 0.05 versus untreated [one-way analysis of variance (ANOVA)]. (B) Editing efficiency in the bone marrow and spleen-derived human CD45+ progeny of repopulating HSPCs, as evaluated by Sanger sequencing and TIDE analysis. The proportion of edited alleles in the input HSPC populations (: HSPCs cultured for 6 days in HSPC medium; : BFU-E; : CFU-GM) is indicated (input). Values shown are means SEM. Each data point represents an individual mouse. (C) Genome editing efficiency in the input populations and in bone marrow and spleen-derived human CD45+ populations edited with the 197, 196, or 115 gRNAs, as evaluated by Sanger sequencing and TIDE analysis. The main events associated with MH-motifs are indicated. Values shown are means SEM (n = 4 mice per group). ***P 0.001, **P 0.01, and *P 0.05 versus input (unpaired t test).

Therapeutic approaches aimed at increasing HbF levels could rely on the down-regulation of nuclear factors involved in -globin silencing. However, knockdown of the transcriptional repressor LRF increases HbF expression but delays erythroid differentiation (28). Here, we used a CRISPR-Cas9 strategy to disrupt the cis regulatory element involved in LRF-mediated fetal globin silencing and mimic the effect of HPFH mutations. By using three different gRNAs targeting the LRF-binding site, we achieved a robust, virtually pancellular HbF reactivation and a concomitant reduction in S-globin levels, recapitulating the phenotype of asymptomatic SCD-HPFH patients (29, 30). Notably, a proportion of HbF >30% in 70% of RBCs has been proposed as the minimal requirement to inhibit HbS polymerization and mitigate the clinical SCD manifestations (30). RBCs derived from edited HSPCs displayed HbF levels sufficient to significantly ameliorate the SCD cell phenotype. It is noteworthy that this approach can potentially be applied also to -thalassemias, where elevated fetal -globin levels could compensate for -globin deficiency.

The development of a selection-free, optimized editing protocol allowed us to obtain a high editing frequency at the LRF-binding site in primary human HSPCs and in HSC-enriched cell populations, which, unexpectedly, showed editing events potentially generated by both NHEJ and MMEJ. However, similarly to the homology-directed repair mechanism (18) [used to correct disease-causing mutations (3133)], the MMEJ repair pathway occurs in actively dividing cells (34). Therefore, we could not exclude that MMEJ might not be efficient in the quiescent repopulating HSCs (24, 25). Xenotransplantation of HSPCs edited using the gRNAs targeting the LRF- or the BCL11A-binding sites demonstrated a high editing efficiency in repopulating HSPCs and no impairment of their multilineage potential. Similarly to recent studies (24, 35), we observed the persistence in vivo of the 13-bp deletion in the 115 region (although at a lower frequency compared to in vitro cultured HSPCs), which is predicted to be mediated by MMEJ. Upon targeting of the BCL11A enhancer, Wu and colleagues (25) observed a stronger reduction in the frequency of editing events possibly due to MMEJ. In our study, upon targeting of the 200 region, some, but not all, deletions associated with MH motifs and potentially generated via MMEJ were detected at a significantly lower frequency in engrafting HSPCs compared to in vitro cultured HSPCs. Together, these studies suggest that, although at a lower frequency compared to in vitro cultured hematopoietic progenitors, MMEJ can occur in vivo in repopulating HSPCs, in which, however, NHEJ is likely the most active repair pathway. However, as MH motifassociated editing events are only computationally predicted to be due to MMEJ, we cannot exclude that a fraction of these events are caused by NHEJ and therefore can occur in repopulating HSPCs.

It is noteworthy that larger deletions typically generated by the 197 edits and associated with an efficient disruption of the LRF-binding sites occur also in vivo. Moreover, even short InDels generated mainly by NHEJ (e.g., 196 gRNA) and detected in repopulating HSPCs are productive in terms of HbF derepression and correction of the SCD cell phenotype. Together, these results show that this strategy can be effective in engrafting HSPCs.

Should the observed editing frequency be confirmed in vivo in patients, this approach would guarantee the efficiency required to achieve clinical benefit in SCD and -thalassemia. The clinical history of allogeneic HSC transplantation for both diseases suggests that a limited fraction of genetically corrected HSCs would be sufficient to achieve a therapeutic benefit given the in vivo selective survival of corrected RBCs or erythroid precursors (3641).

Disrupting either the LRF- or the BCL11A-binding site in the HBG promoters induced significant HbF production. Given the independent role of LRF and BCL11A in -globin repression (28), combined strategies aimed at evicting simultaneously both repressors from the -globin promoters could have an additive effect on HbF reactivation. Albeit a Cas9-nucleasebased strategy targeting both the 115 and 200 regions would probably trigger the deletion of the 115-to-200 intervening sequence [that would be detrimental for promoter activity; (42)], this study paves the way for the use of novel DSB-free editing strategies [e.g., base editing (43)] to simultaneously disrupt both LRF and BCL11A repressor binding sites in the -globin promoters.

Overall, our study provides proof of concept for a novel approach to treat SCD by targeting a repressor binding site in the -globin promoters to induce derepression of fetal Hb and a concomitant decrease in HbS synthesis. The same strategy could be beneficial also in the case of -thalassemia, potentially providing a more economical gene therapy approach compared to the use of LV vectors to deliver a functional -globin gene. LV manufacturing is complex and very expensive (44). Our genome editing approach requires the delivery of RNA/protein reagents that might be less expensive than LV production and thus would allow the broader use of gene therapy for -hemoglobinopathies.

Clinical translation of this genome editing approach requires the development of nontoxic large-scale transfection protocols based on clinical-grade reagents and demonstration of precise editing in a number of HSPCs at least comparable to the efficacious doses predicted by allogeneic transplantation data (i.e., 2 106 to 3 106 HSPCs/kg).

We used CRISPOR (45) to design gRNAs targeting the 200 and 158 regions of the HBG promoters (Table 1). For gRNA expression in erythroid cell lines, oligonucleotide duplexes containing the gRNA protospacers were ligated into Bbs Idigested MA128 plasmid (provided by M. Amendola, Genethon, France). For RNP delivery to HSPCs, we used chemically modified synthetic single gRNAs harboring 2-O-methyl analogs and 3-phosphorothioate nonhydrolyzable linkages at the first three 5 and 3 nucleotides (Synthego) at a concentration of 180 M. Two-part cr:tracrRNA gRNAs were composed of a tracrRNA (IDT) and a custom crRNA (IDT) assembled in equimolar concentrations to produce a 180 M duplex (Table 1).

Protospacer adjacent motifs (PAMs) are highlighted in bold.

K562 were maintained in RPMI 1640 (Lonza) containing glutamine and supplemented with 10% fetal bovine serum (Lonza), Hepes (Life Technologies), sodium pyruvate (Life Technologies), and penicillin and streptomycin (Life Technologies). HUDEP-2 cells (46) were cultured and differentiated, as previously described (47). Flow cytometric analysis of CD36, CD71, and GYPA surface markers and a standard May-Grnwald Giemsa staining were performed to monitor erythroid differentiation.

K562 and HUDEP-2 cells were transfected with 4 g of a Cas9-GFPexpressing plasmid (pMJ920, Addgene) and 0.8 g (K562) and 1.6 g (HUDEP-2) of gRNA-containing plasmid. We used AMAXA Cell Line Nucleofector Kit V (VCA-1003) and U-16 and L-29 programs (Nucleofector II) for K562 and HUDEP-2, respectively. GFP+ HUDEP-2 cells were sorted using SH800 Cell Sorter (Sony Biotechnology).

We obtained human cord blood (CB) CD34+ HSPCs from healthy donors. CB samples eligible for research purposes were obtained because of a convention with the CB bank of Saint Louis Hospital (Paris, France). Human adult SCD CD34+ HSPCs were isolated from Plerixafor mobilized SCD patients (NCT 02212535 clinical trial, Necker Hospital, Paris, France). We obtained granulocyte colony-stimulating factor (G-CSF)mobilized adult HSPCs from healthy donors. Written informed consent was obtained from all adult subjects. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the regional investigational review board (reference: DC 2014-2272, CPP Ile-de-France II Hpital Necker-Enfants malades). HSPCs were purified by immunomagnetic selection with AutoMACS (Miltenyi Biotec) after immunostaining with the CD34 MicroBead Kit (Miltenyi Biotec).

Forty-eight hours before transfection, CD34+ cells (106 cells/ml) were thawed and cultured in the HSPC medium containing StemSpan (STEMCELL Technologies) supplemented with penicillin/streptomycin (Gibco), 250 nM StemRegenin1 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): stem cell factor (SCF) (300 ng/ml), Flt-3L (300 ng/ml), thrombopoietin (TPO) (100 ng/ml), and interleukin-3 (IL-3) (60 ng/ml).

gRNAs were assembled at room temperature with a 90 M Cas9-GFP protein (provided by De Cian) in RNP complexes using a ratio of 2:1 (gRNA:Cas9). Human CD34+ cells (1 106 to 2 106) were transfected with RNP particles using the P3 Primary Cell 4D-Nucleofector X Kit S or L (Lonza), respectively, and the AMAXA 4D CA137 program (Lonza) together with 90 M transfection enhancer (IDT), unless otherwise stated.

Transfected human HSPCs were differentiated into mature RBCs using a three-step protocol (48). From day 0 to day 6, cells were grown in a basal erythroid medium supplemented with the following recombinant human cytokines: SCF (100 ng/ml; PeproTech), IL-3 (5 ng/ml; PeproTech), EPO Eprex (3 IU/ml; Janssen-Cilag), and 106 M hydrocortisone (Sigma). From day 6 to day 9, cells were cultured onto a layer of murine stromal MS-5 cells in a basal erythroid medium supplemented with EPO Eprex (3 IU/ml). Last, from day 9 to day 19, cells were cultured on a layer of MS-5 cells in a basal erythroid medium without cytokines. Erythroid differentiation was monitored by May Grnwald-Giemsa staining; flow cytometric analysis of CD36, CD71, and GYPA erythroid surface markers; and DRAQ5 staining of nucleated cells.

Healthy donor CB-derived CD34+ HSPCs (106) were transfected as described above and plated at a concentration of 500,000/ml in StemSpan (STEMCELL Technologies) supplemented with penicillin/streptomycin (Gibco), 250 nM StemRegenin1 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): SCF (300 ng/ml), Flt-3L (300 ng/ml), TPO (100 ng/ml), and IL-3 (60 ng/ml). Eighteen hours after transfection, cells were stained with antibodies recognizing CD34 [CD34 phycoerythrin (PE)Cy7, 348811; BD Pharmingen], CD133 (CD133 PE, 130-113-748, Miltenyi Biotec), and CD90 (CD90 PE-Cy5, 348811, BD Pharmingen). Cells were sorted using FACSAria II (BD Biosciences). Sorted and unsorted populations were cultured at a concentration of 5 105/ml in a cytokine-enriched medium (described above) for 4 days before collection for DNA extraction.

The number of hematopoietic progenitors was evaluated by clonal CFC assay. HSPCs were plated at a concentration of 1 103 cells/ml in a methylcellulose-containing medium (GFH4435, STEMCELL Technologies) under conditions supporting erythroid and granulomonocytic differentiation. BFU-E and CFU-GM colonies were scored after 14 days. BFU-Es and CFU-GMs were randomly picked and collected as bulk populations (containing at least 25 colonies) or as individual colonies (35 to 45 colonies per sample) to evaluate genome editing efficiency.

Genome editing was analyzed in HUDEP-2 cells at days 0 and 9 of erythroid differentiation and in CB and adult mobilized HSPC-derived erythroid cells at days 6 and 14 of erythroid differentiation, respectively. Genomic DNA was extracted from control and edited cells using the PureLink Genomic DNA Mini Kit (Life Technologies), Quick-DNA/RNA Miniprep (ZYMO Research), or DNA Extract All Reagents Kit (Thermo Fisher Scientific) following the manufacturers instructions. To evaluate NHEJ efficiency at gRNA target sites, we performed PCR followed by Sanger sequencing and TIDE analysis (tracking of InDels by decomposition) (49) or ICE CRISPR Analysis Tool (Synthego) (Table 2) (50).

F, forward primer; R, reverse primer.

Digital droplet PCR was performed using EvaGreen mix (Bio-Rad) to quantify the frequency of the 4.9-kb deletion. Short (~1 min) elongation time allowed the PCR amplification of the genomic region harboring the deletion. Control primers annealing to a genomic region on the same chromosome (chr 11) were used as DNA loading control (Table 3).

Following PCR amplification of the target sequences with the Phusion High-Fidelity polymerase with GC Buffer (New England BioLabs), amplicons were purified using Ampure XP beads (Beckman Coulter). Illumina-compatible barcoded DNA amplicon libraries were prepared using the TruSeq DNA PCR-Free kit (Illumina). PCR amplification was then performed using 1 ng of double-stranded ligation product and Kapa Taq polymerase reagents (KAPA HiFi HotStart ReadyMix PCR Kit, Kapa Biosystems). After a purification step using Ampure XP beads (Beckman Coulter), libraries were pooled and sequenced using Illumina HiSeq2500 (paired-end sequencing 130 130 bases) (Table 4).

For the on-target sites, read pairs were assembled using FLASH. We used a custom python tool suite to count and characterize InDels that were classified in different types: 1-bp deletions, >1-bp deletions non-MH (not associated with MH motifs), >1-bp deletions MH (associated with MH motifs), 1-bp insertions, and >1-bp insertions and substitutions. A tunable window around the cleavage site (typically of 10 bp) was defined to minimize false-positive InDels, and comparison between treated and control samples was used to call InDels due to treatment versus sequencing errors. For the off-target sites, targeted deep sequencing data were analyzed using CRISPRESSO (51).

Human embryonic kidney (HEK) 293T/17 cells (2.5 105) were transfected with 500 ng of a SpCas9-expressing plasmid, together with 250 ng of each single-guide RNAcoding plasmid or an empty pUC19 vector (background control), 10 pmol of the bait dsODN (designed according to the original GUIDE-seq protocol), and 50 ng of a pEGFP-IRES-Puro plasmid, expressing both enhanced GFP (EGFP) and the puromycin resistance genes. One day after transfection, cells were replated and selected with puromycin (1 g/ml) for 48 hours to enrich for transfected cells. Cells were then collected, and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and sheared to an average length of 500 bp with the Bioruptor Pico Sonication System (Diagenode). Library preparation was performed using the original adapters and primers according to previous work (27). Libraries were sequenced with a MiSeq sequencing system (Illumina) using an Illumina MiSeq Reagent kit V2-300 cycles (2 150-bp paired-end). Raw sequencing data (FASTQ files) were analyzed using the GUIDE-seq computational pipeline (52). Identified sites were considered bona fide off-targets if a maximum of seven mismatches against the on-target were present and if they were absent in the background control. The GUIDE-seq datasets are available in the BioProject repository under the accession number PRJNA531587.

Total RNA was extracted from differentiated HUDEP-2 (day 9) and primary mature SCD erythroblasts (day 13) using an RNeasy Micro kit (Qiagen), following the manufacturers instructions. Mature transcripts were reverse-transcribed using SuperScript First-Strand Synthesis System for RT-qPCR (Invitrogen) with oligo(dT) primers. RT-qPCR was performed using an iTaq Universal SYBR Green master mix (Bio-Rad) and a Viia7 Real-Time PCR system (Thermo Fisher Scientific) (Table 5).

RP-HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and the LC Solution software (Shimadzu). Globin chains were separated by HPLC using a 250 mm 4.6 mm, 3.6-m Aeris Widepore column (Phenomenex). Samples were eluted with a gradient mixture of solution A (water/acetonitrile/trifluoroacetic acid, 95:5:0.1) and solution B (water/acetonitrile/trifluoroacetic acid, 5:95:0.1). The absorbance was measured at 220 nm.

CE-HPLC analysis was performed using a NexeraX2 SIL-30 AC chromatograph and the LC Solution software (Shimadzu). Hb tetramers were separated by HPLC using two cation-exchange columns (PolyCAT A, PolyLC, Columbia, MD). Samples were eluted with a gradient mixture of solution A [20 mM Bis-Tris and 2 mM KCN (pH 6.5)] and solution B [20 mM Bis-Tris, 2 mM KCN, and 250 mM NaCl (pH 6.8)]. The absorbance was measured at 415 nm. The calculation of total Hb levels was performed by integration of the areas under the Hb peaks followed by comparison with a standard Hb control (Lyphochek Hemoglobin A2 Control, Bio-Rad).

Differentiated HUDEP-2 cells were fixed and permeabilized using BD Cytofix/Cytoperm solution (BD Pharmingen) and stained with an antibody recognizing HbF [an allophycocyanin (APC)conjugated anti-HbF antibody, MHF05, Life Technologies or a fluorescein isothiocyanate (FITC)conjugated anti-HbF antibody, 552829, BD Pharmingen]. HSPC-derived RBCs or erythroblasts were fixed in cold 0.05% glutaraldehyde and permeabilized using 0.1% Triton X-100. After incubation with Fcr Blocking Reagent (Miltenyi Biotec), cells were stained with an FITC-conjugated anti-HbF antibody (552829, BD Pharmingen).

Flow cytometric analysis of CD36, CD71, and GYPA erythroid surface markers was performed using a V450-conjugated anti-CD36 antibody (561535, BD Horizon), an FITC-conjugated anti-CD71 antibody (555536, BD Pharmingen), and a PE-Cy7conjugated anti-GYPA antibody (563666, BD Pharmingen). We used the nuclear dye DRAQ5 (eBioscience) to evaluate the proportion of enucleated RBCs.

To determine genome editing efficiency in erythroid subpopulations, cells were labeled with a PE-Cy7conjugated anti-GYPA antibody (563666, BD Pharmingen) and an FITC-conjugated anti-HbF antibody (552829, BD Pharmingen), as described above. GYPA+ cells were sorted on the basis of HbF expression using FACSAria II (BD Biosciences).

Flow cytometry analyses were performed using Fortessa X20 (BD Biosciences) or Gallios (Beckman Coulter) flow cytometers. Data were analyzed using the Kaluza software (Beckman Coulter) or the FlowJo software (BD Biosciences).

ChIP experiments to detect H3K27Ac were performed as previously described (53). After 5 days of differentiation, 197 and AAVS1 HUDEP-2 bulk populations were collected for ChIP assays. Briefly, chromatin was cross-linked for 10 min at room temperature with 1% formaldehyde-containing medium. Nuclear extracts were sonicated using the Bioruptor Pico Sonication System (Diagenode). Chromatin obtained from 2 106 cells was immunoprecipitated at 4C overnight using an antibody (1 g per 106 cells) against H3K27Ac (ab4729, Abcam) or a control immunoglobulin G (sc-2025, Santa Cruz Biotechnology). Chromatin cross-linking was reversed at 65C for at least 4 hours, and DNA was purified using the QIAquick PCR purification kit (Qiagen). We used quantitative SYBR Green PCR (Applied Biosystems) and the Viia7 Real-Time PCR System (Thermo Fisher Scientific) to evaluate H3K27Ac enrichment at different genomic loci (Table 6). ChIP experiments to detect LRF were performed as previously described (11) in 197- and AAVS1-edited K562 bulk populations (Table 7).

HSPC-derived SCD RBCs were exposed to an oxygen-deprived atmosphere (0% O2), and the time course of sickling was monitored in real time by video microscopy, capturing images every 20 min for at least 80 min using an AxioObserver Z1 microscope (Zeiss) and a 40 objective. Images of the same fields were taken throughout all stages and processed with ImageJ to determine the percentage of nonsickled RBCs per field of acquisition in the total RBC population. Cells (~300 to 3300) were counted per condition (1500 cells on average).

Nonobese diabetic severe combined immunodeficiency gamma (NSG) mice (NOD.CgPrkdcscid Il2rgtm1Wj/SzJ, Charles River Laboratories, St Germain sur lArbresle, France) were housed in a specific pathogenfree facility. Mice at 6 to 8 weeks of age were conditioned with busulfan (Sigma, St. Louis, MO, USA) injected intraperitoneally (25 mg/kg body weight/day) 24, 48, and 72 hours before transplantation. Control or edited mobilized healthy donor CD34+ cells (106 cells per mouse) were transplanted into NSG mice via retro-orbital sinus injection. Neomycin and acid water were added in the water bottle. At 16 weeks after transplantation, NSG recipients were sacrificed. Cells were harvested from femur bone marrow, thymus, and spleen; stained with antibodies against murine or human surface markers [murine CD45, BD Biosciences, Franklin Lakes, NJ, USA; human CD45, Miltenyi Biotec, Bergisch Gladbach, Germany; human CD3, Miltenyi Biotec, Bergisch Gladbach, Germany; human CD14, BD Biosciences, Franklin Lakes, NJ, USA; human CD15, Beckman Coulter, Brea, CA, USA; human CD19, Sony Biotechnologies, San Jose, CA, USA; human CD235a (CD235a-APC), BD Pharmingen]; and analyzed by flow cytometry using a Gallios analyzer and the Kaluza software (Beckman Coulter, Brea, CA, USA). All experiments and procedures were performed in compliance with the French Ministry of Agricultures regulations on animal experiments and were approved by the regional Animal Care and Use Committee (APAFIS#2101-2015090411495178 v4).

Paired t tests were performed to compare genome editing efficiencies in erythroid subpopulations sorted based on HbF expression. Unpaired t tests were performed for all the other analyses. Statistical analyses were carried out using Prism4 software (GraphPad). We used the Kruskal-Wallis test to compare frequency of deletion generated at each nucleotide by the different gRNAs. The threshold for statistical significance was set to P < 0.05.

Acknowledgments: We thank R. Kurita and Y. Nakamura for contributing the HUDEP-2 cell line, L. Douay for the erythroid differentiation protocol, G. Pavani for the optimization of editing protocol in HSPCs, B. Wienert for providing assistance and protocol for the LRF ChIP, O. Alibeau and C. Bole for the DNA sequencing, E. Brunet for the discussion, and E. Duvernois-Berthet for the script used for InDel characterization. Funding: This work was supported by grants from the European Research Council (ERC-2015-AdG, GENEFORCURE), the Agence Nationale de la Recherche (ANR-16-CE18-0004, ANR-11-INBS-0014-TEFOR, ANR-17-CE13-0016-i-MMEJ, and ANR-10-IAHU-01 Investissements davenir program), the Paris Ile-de-France Region under DIM Thrapie gnique initiative, and Genopole (CHAIRE JUNIOR FONDAGEN). Author contributions: L.W. and G.F. designed and conducted the experiments and wrote the paper. T.F., G.H., A.Ca., C.W., V.M., and A.Ch. designed and conducted the experiments. C.M. analyzed off-target NGS data. A.D.C. provided reagents. F.M., M.A., I.A.-S., A.Ce., W.E.N., J.-P.C., C.G., and M.C. contributed to the design of the experimental strategy. A.M. conceived the study, designed the experiments, and wrote the paper. Competing interests: A.M. and L.W. are inventors on a patent application related to this work filed by INSERM (PCT/EP2019/074131, 10 September 2019). The authors declare that they have no other competing interests. Data and materials availability: The GUIDE-seq datasets are available in the BioProject repository under the accession number PRJNA531587. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Editing a -globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype - Science Advances